Determination of Neomycin B and Impurities Using HPAE-IPAD

Importance of the topic

The accurate determination of Neomycin B and its related impurities is critical for quality control of aminoglycoside antibiotics. Neomycin B is the most potent component of a complex fermentation product with multiple structurally similar impurities. Conventional UV or refractive detectors lack sensitivity and selectivity for these nonchromophoric analytes. High performance anion exchange chromatography with integrated pulsed amperometric detection delivers the chemical specificity and low detection limits required for trace analysis in pharmaceutical formulations.

Objectives and overview

This study describes the development and validation of an automated HPAE-IPAD method to quantify Neomycin B and its impurities. Major goals included eliminating manual mobile phase preparation variability by using an eluent generator, improving retention time reproducibility with a carbonate trap, and applying the method to a complex topical ointment. Performance was evaluated following pharmacopeial guidelines for linearity, sensitivity, precision, accuracy, robustness, and system suitability.

Methodology and instrumentation

The analytical setup comprised a CarboPac PA1 column with PA1 guard, an EG50 eluent generator producing 2.40 mM KOH, a continuously regenerated anion trap column, and an ED50 pulsed amperometric detector fitted with disposable gold working electrodes. The eluent generator prepared carbonate free hydroxide mobile phase in situ. Separations were conducted at 0.5 mL per minute, 30 C, with 20 microliter injections. Neosporin topical ointment was extracted by heating with water, vortex mixing, centrifugation, and gravimetric dilution prior to analysis.

Main results and discussion

Baseline resolution of Neomycin B from primary impurities and matrix components was achieved in a 15 minute run. Peak area response was linear from 0.011 to 20 micromolar (0.21 to 400 pmol) with r2 of 0.9946. The limit of detection was 0.011 micromolar and the limit of quantitation was 0.036 micromolar. Intra and interday retention time relative standard deviation was 0.64 percent and peak area RSD was 1.33 percent over ten days and 822 injections. Spike recoveries from ointment extracts averaged 99.6 percent with 2.5 percent RSD. Measured Neomycin B content in Neosporin was 4.15 mg per gram, consistent with product specifications. The method exceeded resolution and sensitivity requirements of USP and EP compendial methods.

Benefits and practical applications

This automated HPAE IPAD workflow eliminates manual mobile phase preparation errors and reduces pump maintenance by handling only water and electrolyte. Disposable gold electrodes provide consistent electrochemical response and require no polishing. The method generates benign waste that can be neutralized before disposal. It is well suited for routine pharmaceutical quality control, stability studies, and impurity profiling of aminoglycoside antibiotics.

Future trends and potential uses

Future developments may include extending the method to other aminoglycosides and carbohydrate based drugs, integration with on line process monitoring, adoption of higher throughput or multiplexed formats, and further miniaturization of electrochemical sensors. Advances in automated eluent generation and electrode technology are expected to enhance trace analysis capabilities in biopharmaceutical manufacturing and research.

Conclusion

The presented HPAE IPAD method with eluent generation and disposable gold working electrodes provides a robust, sensitive, and reproducible approach for Neomycin B and impurity determination. It meets stringent pharmacopeial criteria and can be readily implemented in pharmaceutical analysis laboratories.

Reference

1. Physicians Desk Reference 44th Edition 1990 Medical Economics Company Oradell NJ
2. Swart EA Romano AH Waksman SA Proc Soc Exp Biol Med 1950 73 376
3. Lemieux RU Kullnig RK Moir RY J Am Chem Soc 1958 80 2237
4. Rinehart KL The Neomycins and Related Antibiotics Chemistry of Microbial Products John Wiley Sons 1964
5. United States Pharmacopeia USP 29 NF 24 Neomycin Sulfate 2006 p1491
6. European Pharmacopoeia Fifth Edition Neomycin Sulfate Section 0197 2005
7. Szunyog J Adams E Roets E Hoogmartens J J Pharm Biomed Anal 2000 23 891-896
8. Polta JA Johnson DC Merkel KE J Chromatogr 1985 324 407-414
9. Adams E Schepers R Roets E Hoogmartens J J Chromatogr A 1996 741 233-240
10. Application Note 66 Determination of Neomycin in Topical Lotions Dionex 1991
11. Application Note 61 Determination of Tobramycin and Impurities Using HPAE-PAD Dionex 2004
12. Hanko VP Rohrer JS J Pharm Biomed Anal 2006 40 1006-1012
13. Cheng J Jandik P Avdalovic N Anal Chem 2003 75 572-579
14. Cheng J Jandik P Avdalovic N J Chromatogr A 2003 997 73-78
15. ICH Q2A Validation of Analytical Procedures 1994
16. ICH Q2B Validation of Analytical Procedures Methodology 1996
17. FDA CDER CBER Guidance Analytical Procedures and Method Validation 2000
18. FDA CDER Reviewer Guidance Validation of Chromatographic Methods 1994
19. FDA CDER Guideline for Submitting Samples and Analytical Data for Methods Validation 1987
20. FDA CDER CVM Guidance for Industry Bioanalytical Method Validation 2001
21. USP 29 NF 24 Validation of Compendial Methods 1225 2006
22. USP 29 NF 24 Chromatography 621 2006
23. ICH Q3AR Impurities in New Drug Substances 2002
24. Dionex Product Manual AAA Direct Amino Acid Analysis System 031481
25. MedlinePlus National Library of Medicine Neomycin 2006
26. United States Pharmacopeia USP 29 NF 24 Neomycin Polymyxin B Sulfates and Bacitracin Zinc Ointment 2006 1503